Decellularized human colorectal cancer matrices polarize macrophages towards an anti-inflammatory phenotype promoting cancer cell invasion via CCL18.

Macrophages are frequently identified in solid tumors, playing important roles in cancer progression. Their remarkable plasticity makes them very sensitive to environmental factors, including the extracellular matrix (ECM). In the present work, we investigated the impact of human colorectal tumor matrices on macrophage polarization and on macrophage-mediated cancer cell invasion. Accordingly, we developed an innovative 3D-organotypic model, based on the decellularization of normal and tumor tissues derived from colorectal cancer patients' surgical resections. Extensive characterization of these scaffolds revealed that DNA and other cell constituents were efficiently removed, while native tissue characteristics, namely major ECM components, architecture and mechanical properties, were preserved. Notably, normal and tumor decellularized matrices distinctly promoted macrophage polarization, with macrophages in tumor matrices differentiating towards an anti-inflammatory M2-like phenotype (higher IL-10, TGF-ß and CCL18 and lower CCR7 and TNF expression). Matrigel invasion assays revealed that tumor ECM-educated macrophages efficiently stimulated cancer cell invasion through a mechanism involving CCL18. Notably, the high expression of this chemokine at the invasive front of human colorectal tumors correlated with advanced tumor staging. Our approach evidences that normal and tumor decellularized matrices constitute excellent scaffolds when trying to recreate complex microenvironments to understand basic mechanisms of disease or therapeutic resistance.

Three-dimensional scaffolds of fetal decellularized hearts exhibit enhanced potential to support cardiac cells in comparison to the adult.

A main challenge in cardiac tissue engineering is the limited data on microenvironmental cues that sustain survival, proliferation and functional proficiency of cardiac cells. The aim of our study was to evaluate the potential of fetal (E18) and adult myocardial extracellular matrix (ECM) to support cardiac cells. Acellular three-dimensional (3D) bioscaffolds were obtained by parallel decellularization of fetal- and adult-heart explants thereby ensuring reliable comparison. Acellular scaffolds retained main constituents of the cardiac ECM including distinctive biochemical and structural meshwork features of the native equivalents. In vitro, fetal and adult ECM-matrices supported 3D culture of heart-derived Sca-1(+) progenitors and of neonatal cardiomyocytes, which migrated toward the center of the scaffold and displayed elongated morphology and excellent viability. At the culture end-point, more Sca-1(+) cells and cardiomyocytes were found adhered and inside fetal bioscaffolds, compared to the adult. Higher repopulation yields of Sca-1(+) cells on fetal ECM relied on ß1-integrin independent mitogenic signals. Sca-1(+) cells on fetal bioscaffolds showed a gene expression profile that anticipates the synthesis of a permissive microenvironment for cardiomyogenesis. Our findings demonstrate the superior potential of the 3D fetal microenvironment to support and instruct cardiac cells. This knowledge should be integrated in the design of next-generation biomimetic materials for heart repair.

Multipotent hematopoietic progenitor cells immortalized by Lhx2 self-renew by a cell nonautonomous mechanism.

OBJECTIVE: Direct molecular and cellular studies of hematopoietic stem cells (HSCs) are hampered by the low levels of HSCs in hematopoietic tissues. To address these issues, we generated immortalized multipotent hematopoietic precursor cell (HPC) lines by expressing the LIM-homeobox gene Lhx2 (previously LH2) in hematopoietic progenitors derived from embryonic stem cells differentiated in vitro. MATERIALS AND METHODS: To validate further the relevance of the HPC lines as a model for normal HSCs, we analyzed in detail the growth requirements of HPC lines in vitro. RESULTS: Lhx2 immortalized the HPC lines by a putatively novel and cell nonautonomous mechanism. Self-renewal of the HPC lines is dependent on functional Lhx2 expression. Most early-acting hematopoiesis-related growth factors show synergistic effects on the HPC lines, whereas late-acting factors do not induce differentiation by themselves. Transforming growth factor-beta(1) is a potent inhibitor of proliferation of the HPC lines. HPC lines form cobblestone areas with high efficiency when seeded onto stromal cell lines, and the cobblestone area-forming cell can be maintained in these cultures for several months. CONCLUSIONS: Our data show that, in many respects, HPC lines are similar to normal hematopoietic progenitor/stem cells on the cellular level, in contrast to most previously described multipotent hematopoietic cell lines. The cell nonautonomous mechanism for immortalization of the HPC lines suggests that Lhx2 regulates, directly or indirectly, soluble mediators involved in self-renewal of the HPC lines.

Expression of the LIM-homeobox gene LH2 generates immortalized steel factor-dependent multipotent hematopoietic precursors.

The genes controlling self-renewal and differentiation in the hematopoietic system are largely unknown. The LIM-homeobox genes are known to be important for asymmetric cell divisions and differentiation of specific cell types and organs. One member of this family, LH2, is expressed in fetal liver at the time of active hematopoiesis. Therefore, we have assessed the function of LH2 during the formation and initial expansion of the hematopoietic system by differentiating LH2-transduced embryonic stem (ES) cells in vitro. This procedure generated multipotent hematopoietic precursor cell (HPC) lines that required Steel factor for growth. HPC lines have been maintained in an undifferentiated state in culture for >7 months. Other growth factors tested efficiently induce terminal differentiation of HPCs into various mature myeloid lineages. Steel factor is also required and acts synergistically with the other growth factors to generate multilineage colonies from the HPCs. These HPC lines express transcription factors that are consistent with an immature progenitor, and the pattern of cell surface marker expression is similar to that of early fetal multipotent hematopoietic progenitors. Collectively, these data suggest that the HPC lines represent an early fetal multipotent hematopoietic progenitor, and suggest a role for LH2 in the control of cell fate decision and/or proliferation in the hematopoietic system.

Short-term vs. sustained inhibition of proximal tubule Na,K-ATPase activity by dopamine: cellular mechanisms.

Dopamine (DA) produces a natriuresis attributed in part to inhibition of Na,K-ATPase activity (NKA) in the proximal tubule (PCT), and impairment in this inhibition has been linked to several forms of hypertension in animals. Here we examined whether the intracellular signaling mechanisms involved are the same in the early and late phases of this phenomenon. DA (1 microM) inhibited NKA similarly after 15 min (by 38%) or 180 min (by 36%) incubation, taken to represent short-term (ST) and sustained (Sd) pump regulation, respectively. Calphostin C, a specific inhibitor of protein kinase C (PKC), completely blocked the ST action of DA on NKA, whereas IP20, a specific inhibitor of protein kinase (PKA), had no effect. In contrast, IP20 completely abolished the Sd (180 min) inhibition by DA, whereas calphostin C had only a partial or variable effect. The DA-1 agonist fenoldopam (which does not activate PKC but increases cAMP) alone failed to inhibit the pump at 180 min (as it does also in the short-term in PCT), suggesting that ST inhibition is required for the Sd effect to occur. Furthermore, PTH1-34, a known ST inhibitor of NKA suppressed the pump at 180 min (by 46%), but unlike in the short-term, this effect was completely prevented by IP20. In contrast, PTH3-34, which does not stimulate adenylyl cyclase or activate PKA, caused only a small (19%) and variable Sd inhibition. In conclusion, short-term inhibition of the PCT pump by dopamine is mediated via PKC, whereas the sustained inhibition requires the PKA pathway in addition to the ongoing PKC-mediated effect.

Studies on the pharmacology of the inward transport of L-DOPA in rat renal tubules.

1. The accumulation of L-DOPA in suspensions of renal tubules obtained from male Wistar rats occurred through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA uptake in renal tubules was as follows: Vmax = 46.3 +/- 2.8 nmol mg-1 protein h-1 and Km = 114.4 (95% confidence limits; 83.8, 156.2) microM (n = 5). The diffusion constant (in nmol-1) of the non-saturable component for the accumulation of L-DOPA was 1.3 (1.1, 1.6; n = 8). 2. The effect of 2,4-dinitrophenol was a marked reduction in the tubular uptake of L-DOPA, with an IC50 value of 12.1 (4.0, 36.9) microM. Cocaine produced a slight (P = 0.08) decrease (22% reduction at 50 microM) in the tubular uptake of L-DOPA. Corticosterone produced a considerable inhibitory effect on the uptake of L-DOPA with an IC50 value of 11.0 (3.6, 33.5) microM. The maximal inhibitory effect of probenecid was a 24% decrease in the uptake of L-DOPA; however, the more selective organic anion transport inhibitor 4,4'-diisothiocyantostilbene-2,2'-disulphonic acid (DIDS) was, found not to affect the tubular uptake of L-DOPA. The organic cation transport inhibitor, cyanine 863, was found to produce a marked decrease in the uptake of L-DOPA (IC50 = 2.02 [1.07, 3.79]). The cyanine derivatives 1,1'-diethyl-2,2'-cyanine (decynium 22) and 1,1'-diethyl-2,4'-cyanine (decynium 24) also potently inhibited L-DOPA uptake with IC50 values (in microM) of 0.63 (0.39, 1.01) and 0.10 (0.08, 0.13), respectively, both compounds were found to be more potent than cyanine 863. 3. The inhibitory effect of decynium 24 (0.2 microM) on the tubular uptake of L-DOPA was dependent on the pH of the incubation medium; at pH = 6.5 the accumulation of L-DOPA was reduced up to 37 +/- 2% of control values, whereas at pH = 7.4 and pH = 8.2 the accumulation of L-DOPA was reduced by 56 +/- 1% and 60 +/- 6%, respectively. Cyanine 863 (2 microM), decynium 22 (1 microM) and decynium 24 (0.2 microM) were found to decrease the Vmax values for the saturable component of L-DOPA uptake without changes in Km values. 4. It is concluded that the tubular uptake of L-DOPA might be promoted through a mechanism which is dependent on the activity of the organic cation transport system.

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: effects of amine precursors on the cell inward transfer and decarboxylation.

1. The present work was designed to examine the interference of L-3,4-dihydroxyphenylalanine (L-DOPA) on the cell inward transport of L-5-hydroxytryptophan (L-5-HTP) and on its decarboxylation by aromatic L-amino acid decarboxylase (AAAD) in rat isolated renal tubules. 2. The accumulation of both L-5-HTP and L-DOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component L-5-HTP and L-DOPA uptake in renal tubules were as follows: L-5-HTP, Vmax = 24.9 +/- 4.5 nmol mg-1 protein h-1 and Km = 121 (95% confidence limits: 75, 193) microM (n = 5); L-DOPA, Vmax = 58.0 +/- 4.3 nmol mg-1 protein h-1 and Km = 135 (97, 188) microM (n = 5). When the saturation curve of L-5-HTP tubular uptake was performed in the presence of L-DOPA (250 microM), the maximal rate of accumulation of L-5-HTP in renal tubules was found to be markedly (P < 0.01) reduced (Vmax = 10.5 +/- 1.7 nmol mg-1 protein h-1, n = 4); this was accompanied by a significant (P < 0.05) increase in Km values (325 [199, 531] microM, n = 4). 3. L-DOPA (50 to 2000 microM) was found to produce a concentration-dependent decrease (38% to 91% reduction) in the tubular uptake of 5-HTP; the Ki value (in microM) of L-DOPA for inhibition of L-5-HTP uptake was found to be 29.1 (13.8, 61.5) (n = 6). 4. At the highest concentration tested the organic anion inhibitor, probenecid (10 microM) produced no significant (P = 0.09) changes in L-5-HTP and L-DOPA uptake (18% and 22% reduction, respectively). The organic cation inhibitor, cyanine 863 (1-ethyl-2-[1,4-dimethyl-2-phenyl-6-pyrimidinylidene)methyl]-quino linium) produced a potent inhibitory effect on the tubular uptake of L-5-HTP (Ki = 212 [35, 1289] nM, n = 8), being slightly less effective against L-DOPA uptake (Ki = 903 [584, 1396] nM, n = 5). The cyanine derivatives 1,1-diethyl-2,4-cyanine (decynium 24) and 1,1-diethyl-2,2-cyanine (decynium 22) potently inhibited the tubular uptake of both L-5-HTP (Ki = 100 [49, 204] and 120 [26, 561] nM, n = 4-6, respectively) and L-DOPA (Ki = 100 [40, 290] and 415 [157, 1094] nM, n = 5, respectively). 5. The Vmax and Km values for AAAD using L-DOPA as the substrate (Vmax = 479.9 +/- 74.0 nmol mg-1 protein h-1; Km = 2380 [1630, 3476] microM; n = 4) were both found to be significantly (P < 0.01) higher than those observed when using L-5-HTP (Vmax = 81.4 +/- 5.2 nmol mg-1 protein h-1, Km = 97 [87, 107] microM, n = 10). The addition of 5 mM L-DOPA to the incubation medium reduced by 30% (P < 0.02) the maximal rate of decarboxylation of L-5-HTP (Vmax = 56.7 +/- 3.1 nmol mg-1 protein h-1, n = 10) and resulted in a significant (P < 0.05) increase in Km values (249 [228, 270] microM, n = 10). 6. The results presented suggest that L-5-HTP and L-DOPA are using the same transporter (most probably, the organic cation transporter) in order to be taken up into renal tubular cells; L-DOPA exerts a competitive type of inhibition upon the tubular uptake and decarboxylation of L-5-HTP. The decrease in the formation of 5-HT as induced by L-DOPA may also depend on a decrease in the rate of its decarboxylation by AAAD.

Antagonistic actions of renal dopamine and 5-hydroxytryptamine: increase in Na+, K(+)-ATPase activity in renal proximal tubules via activation of 5-HT1A receptors.

1. 5-Hydroxytryptamine (5-HT) is antinatriuretic. Since this effect of 5-HT is not accomplished by changes in glomerular haemodynamics, we have examined in this study whether 5-HT may influence sodium excretion by affecting the Na+, K(+)-ATPase activity in renal cortical tubules. 2. Na+, K(+)-ATPase activity was determined as the rate of [32P]-ATP hydrolysis in renal cortical tubules in suspension. Basal Na+, K(+)-ATPase activity in renal tubules was 4.8 +/- 0.4 mumol Pi mg-1 protein h-1 (n = 8). The 5-HT1A receptor agonist, (+/-)-8-hydroxy-2-(di-n-propylamino) tetraline (8-OH-DPAT) (10 to 3000 nM) induced a concentration-dependent increase (P < 0.05) in Na+, K(+)-ATPase activity with an EC50 value of 355 nM (95% confidence limits: 178, 708). Maximal stimulation elicited by 3000 nM of 8-OH-DPAT was antagonized by the selective 5-HT1A receptor antagonist, (+)-WAY 100135 10 to 1000 nM) with an IC50 value of 20 nM (14, 29); 0.3 microM (+)-WAY 100135 completely abolished (P < 0.01) the stimulatory effect of 8-OH-DPAT. The stimulatory effect of 8-OH-DPAT was found to be time-dependent (15 +/- 2% and 66 +/- 7% increase at 2.5 and 5.0 min, respectively). The 5-HT2 receptor agonist alpha-methyl-5-HT (100 to 3000 nM) did not induce any significant changes in Na+, K(+)-ATPase activity (5.0 +/- 1.5 mumol Pi mg-1 protein h-1; n = 4). 3. The stimulatory effect 8-OH-DPAT was absent when homogenates were used. Stimulation occurred at a Vmax concentration (70 mM) of sodium supporting the notion that stimulation occurs independently of increasing sodium permeability. 4. The inhibitory effect of dopamine (P < 0.05) on Na+, K(+)-ATPase activity was blunted by co-incubation with 8-OH-DPAT (0.5 microM). 5. It is concluded that activation of 5-HT1A receptors increases Na+, K(+)-ATPase activity in renal cortical tubules; this effect may represent an important cellular mechanism, at the tubule level, responsible for the antinatriuretic effect of 5-HT.

Assessment of renal dopaminergic system activity during cyclosporine A administration in the rat.

1. Administration of cyclosporine A (CsA; 50 mg kg-1 day-1, s.c.) for 14 days produced an increase in both systolic (SBP) and diastolic (DBP) blood pressure by 60 and 25 mmHg, respectively. The urinary excretion of dopamine, DOPAC and HVA was reduced from day 5-6 of CsA administration onwards (dopamine from 19 to 46%, DOPAC from 16 to 48%; HVA from 18 to 42%). In vehicle-treated rats, the urinary excretion of dopamine and DOPAC increased (from 7 to 60%) from day 5 onwards; by contrast, the urinary excretion of HVA was reduced (from 27 to 60%) during the second week. 2. No significant difference was observed between the Vmax and Km values of renal aromatic L-amino acid decarboxylase (AAAD) in rats treated with CsA for 7 and 14 days or with vehicle. 3. Km and Vmax of monoamine oxidase types A and B did not differ significantly between rats treated with CsA for 7 and 14 days or with vehicle. 4. Maximal catechol-O-methyltransferase activity (Vmax) in homogenates of renal tissues obtained from rats treated with CsA for 7 or 14 days was significantly higher than that in vehicle-treated rats; Km (22.3 +/- 1.5 microM) values for COMT did not differ between the three groups of rats. 5. The accumulation of newly-formed dopamine and DOPAC in cortical tissues of rats treated with CsA for 14 days was three to four times higher than in controls. The outflow of both dopamine and DOPAC declined progressively with time and reflected the amine and amine metabolite tissue contents. No significant difference was observed between the DOPAC/dopamine ratios in the perifusate of renal tissues obtained from CsA- and vehicle-treated rats. In addition, no significant differences were observed in k values or in the slope of decline of both DA and DOPAC between experiments performed with CsA and vehicle-treated animals. 6. The Vmax for the saturable component of L-3,4-dihydroxyphenylalanine (L-DOPA) uptake in renal tubules from rats treated with CsA was twice that of vehicle-treated animals. Km in CsA- and vehicle-treated rats did not differ. 7. The decrease in the urinary excretion of sodium and an increase in blood pressure during CsA treatment was accompanied by a reduction in daily urinary excretion of dopamine. This appears to result from a reduction in the amount of L-DOPA made available to the kidney and does not involve changes in tubular AAAD, the availability of dopamine to leave the renal cells and dopamine metabolism. The enhanced ability of the renal tissues of CsA-treated animals to synthesize dopamine, when exogenous L-DOPA is provided, results from an enhanced activity of the uptake process of L-DOPA in renal tubular cells.

Studies on the nature of the antagonistic actions of dopamine and 5-hydroxytryptamine in renal tissues.

The present work examines the possibility of whether the reciprocal effects of dopamine (DA) and 5-hydroxytryptamine (5-HT) are only dependent on the antagonistic nature of the signal resulting from the activation of their specific receptors or may also result from a competitive type of inhibition at different levels of the synthetic and metabolic pathways shared by DA and 5-HT. Studies performed in isolated proximal convoluted tubules (PCT) have shown that L-5-HTP and L-DOPA use the same transporter in order to be taken up into the cell and both L-DOPA and L-5-HTP exert a competitive type of inhibition upon their cellular uptake. The decrease in the formation of 5-HT in isolated PCT induced by L-DOPA reflects most probably a reduction in the intracellular availability of L-5-HTP. However, in experiments conducted in homogenates of PCT L-DOPA was found to be a better substrate for AAAD than L-5-HTP. Apart from sharing a common synthetic pathway, DA and 5-HT also share a common metabolic pathway; type A monoamine oxidase (MAO-A), the predominant form of MAO in rat renal tissues, converts DA into 3,4-dihydroxyphenylacetic acid (DOPAC) and 5-HT into 5-hydroxyindolacetic acid (5-HIAA). However, in contrast to 5-HT, DA can be metabolized by MAO-B and catechol-O-methyltransferase. Inhibition of MAO-A was found to produce a 2-fold increase in the urinary excretion of 5-HT; this increase in the urinary excretion of 5-HT was accompanied by an unexpected reduction in the urinary excretion of DA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell inward transport of L-DOPA and 3-O-methyl-L-DOPA in rat renal tubules.

1. The present study has determined the kinetics of the uptake of L-3,4-dihydroxyphenylalanine (L-DOPA) and 3-O-methyl-L-DOPA (3-OMDOPA) in rat renal tubules and examined the effect of 3-OMDOPA on the inward transport of L-DOPA and on its conversion into dopamine in kidney homogenates. 2. The accumulation of both L-DOPA and 3-OMDOPA in renal tubules was found to occur through non-saturable and saturable mechanisms. The kinetics of the saturable component of L-DOPA and 3-OMDOPA uptake in renal tubules were as follows: L-DOPA, Vmax = 11.1 nmol mg-1 protein h-1 and Km = 216 microM (n = 6); 3-OMDOPA, Vmax = 8.1 nmol mg-1 protein h-1 and Km = 231 microM (n = 5). The diffusion constant of the non-saturable component for the accumulation of L-DOPA and 3-OMDOPA was 0.0010 and 0.0014 mumol-1, respectively. 3. 3-OMDOPA (100 to 2000 microM) was found to produce a concentration-dependent decrease (29% to 81% reduction) of the saturable component of the tubular uptake of L-DOPA; the Ki value of 3-OMDOPA for inhibition of L-DOPA uptake was found to be 181 microM (n = 5). The accumulation of L-DOPA obtained in experiments conducted at 4 degrees C was not affected by 3-OMDOPA. 4. In experiments conducted in kidney homogenates only L-DOPA (10 to 5000 microM) was found to be decarboxylated. The Vmax and Km values for aromatic L-amino acid decarboxylase determined in the absence of 3-OMDOPA (Vmax = 14.1 nmol mg-1 protein h-1; Km =62 MicroM) were not significantly different from those observed when the decarboxylation of L-DOPA was carried out in the presence of 1000 MicroM 3-OMDOPA (Vmax = 15.7 nmol mg-1 protein h-1; Km = 68 MicroM).5. It is concluded that the tubular uptake of both L-DOPA and 3-OMDOPA occur through nonsaturable and saturable mechanisms; only the saturable tubular uptake of L-DOPA was found to be inhibited by 3-OMDOPA. It is further shown that 3-OMDOPA neither undergoes decarboxylation into 3-MT nor affects the decarboxylation of L-DOPA.